Characterization of microsatellite markers for Sycoscapter nonpollinating fig wasps

We isolated 18 microsatellites from Sycoscapter australis, a nonpollinating fig wasp that develops in figs of Ficus macrophylla, and assessed their variability in 20 wasps. We further optimized nine of these loci for use in three other Sycoscapter species that develop in Ficus rubiginosa figs and assessed their variability in 47-140 wasps per species. These are the first microsatellites developed for nonpollinating fig wasps and show sufficient polymorphism to become important tools in evolutionary and genetical studies of Sycoscapter wasps.

Rapid radiation in spiny lobsters (Palinurus spp) as revealed by classic and ABC methods using mtDNA and microsatellite data

Background: Molecular tools may help to uncover closely related and still diverging species from a wide variety of taxa and provide insight into the mechanisms, pace and geography of marine speciation. There is a certain controversy on the phylogeography and speciation modes of species-groups with an Eastern Atlantic-Western Indian Ocean distribution, with previous studies suggesting that older events (Miocene) and/or more recent (Pleistocene) oceanographic processes could have influenced the phylogeny of marine taxa. The spiny lobster genus Palinurus allows for testing among speciation hypotheses, since it has a particular distribution with two groups of three species each in the Northeastern Atlantic (P. elephas, P. mauritanicus and P. charlestoni) and Southeastern Atlantic and Southwestern Indian Oceans (P. gilchristi, P. delagoae and P. barbarae). In the present study, we obtain a more complete understanding of the phylogenetic relationships among these species through a combined dataset with both nuclear and mitochondrial markers, by testing alternative hypotheses on both the mutation rate and tree topology under the recently developed approximate Bayesian computation (ABC) methods. Results: Our analyses support a North-to-South speciation pattern in Palinurus with all the South-African species forming a monophyletic clade nested within the Northern Hemisphere species. Coalescent-based ABC methods allowed us to reject the previously proposed hypothesis of a Middle Miocene speciation event related with the closure of the Tethyan Seaway. Instead, divergence times obtained for Palinurus species using the combined mtDNA-microsatellite dataset and standard mutation rates for mtDNA agree with known glaciation-related processes occurring during the last 2 my. Conclusion: The Palinurus speciation pattern is a typical example of a series of rapid speciation events occurring within a group, with very short branches separating different species. Our results support the hypothesis that recent climate change-related oceanographic processes have influenced the phylogeny of marine taxa, with most Palinurus species originating during the last two million years. The present study highlights the value of new coalescent-based statistical methods such as ABC for testing different speciation hypotheses using molecular data.

Characterization of microsatellite loci for the endangered cactus Echinocactus grusonii, and their cross-species utilization

Echinocactus grusonii is common in trade but critically endangered in its natural habitat. With the ultimate aim of developing a certification scheme to aid in the conservation of this species, we have isolated E. grusonii microsatellites from a nonenriched library. Fifty-seven sequences contained a microsatellite array, of which 12 were polymorphic among 30 individuals from a single wild population. All 12 microsatellite primer pairs amplified product in one or more species in a screen of 27 other cactus species.

Characterization of microsatellite loci for the critically endangered cactus Ariocarpus bravoanus

Ariocarpus bravoanus is common in trade but critically endangered in its natural habitat. With the ultimate aim of developing a certification scheme to aid in the conservation of this species, we have isolated A. bravoanus microsatellites from a nonenriched library. Fifty-four sequences contained a microsatellite array, of which eight were polymorphic among 23 individuals, 20 from one population and three plants from trade.

A note on the accuracy of PAC-likelihood inference with microsatellite data

Stephens and Donnelly have introduced a simple yet powerful importance sampling scheme for computing the likelihood in population genetic models. Fundamental to the method is an approximation to the conditional probability of the allelic type of an additional gene, given those currently in the sample. As noted by Li and Stephens, the product of these conditional probabilities for a sequence of draws that gives the frequency of allelic types in a sample is an approximation to the likelihood, and can be used directly in inference. The aim of this note is to demonstrate the high level of accuracy of "product of approximate conditionals" (PAC) likelihood when used with microsatellite data. Results obtained on simulated microsatellite data show that this strategy leads to a negligible bias over a wide range of the scaled mutation parameter theta. Furthermore, the sampling variance of likelihood estimates as well as the computation time are lower than that obtained with importance sampling on the whole range of theta. It follows that this approach represents an efficient substitute to IS algorithms in computer intensive (e.g. MCMC) inference methods in population genetics. (c) 2006 Elsevier Inc. All rights reserved.

Microsatellite primers for Ficus racemosa and Ficus rubiginosa

We developed 11 polymorphic microsatellite loci each for the figs Ficus (Sycomorus) racemosa and Ficus (Urostigma) rubiginosa from AG- and TG-enriched genomic libraries. These 22 loci were investigated for cross-species amplification and polymorphism in 17–21 F. racemosa and 16–24 F. rubiginosa individuals from Townsville, Australia. Observed heterozygosities range from 0.12 to 0.90 in F. racemosa and from 0.25 to 1.0 in F. rubiginosa.

Characterization and PCR multiplexing of polymorphic microsatellite loci in cashew (Anacardium occidentale L.) and their cross-species utilization

Cashew (Anacardium occidentale L.) is the most economically important tropical nut crop in the world, and yet there are no sequence tagged site (STS) markers available for its study. Here we use an automated, high-throughput system to isolate cashew microsatellites from a non-enriched genomic library blotted onto membranes at high density for screening. Sixty-five sequences contained a microsatellite array, of which 21 proved polymorphic among a closely related seed garden population of 49 genotypes. Twelve markers were suitable for multiplex analysis. Of these, 10 amplified in all three related tropical tree species tested: Anacardium microcarpum, Anacardium pumilum and Anacardium nanum.

Allelic size standards and reference genotypes to unify international cocoa (Theobroma cacao L.) microsatellite data

Standardisation of microsatellite allele profiles between laboratories is of fundamental importance to the transferability of genetic fingerprint data and the identification of clonal individuals held at multiple sites. Here we describe two methods of standardisation applied to the microsatellite fingerprinting of 429 Theobroma cacao L. trees representing 345 accessions held in the worlds largest Cocoa Intermediate Quarantine facility: the use of a partial allelic ladder through the production of 46 cloned and sequenced allelic standards (AJ748464 to AJ48509), and the use of standard genotypes selected to display a diverse allelic range. Until now a lack of accurate and transferable identification information has impeded efforts to genetically improve the cocoa crop. To address this need, a global initiative to fingerprint all international cocoa germplasm collections using a common set of 15 microsatellite markers is in progress. Data reported here have been deposited with the International Cocoa Germplasm Database and form the basis of a searchable resource for clonal identification. To our knowledge, this is the first quarantine facility to be completely genotyped using microsatellite markers for the purpose of quality control and clonal identification. Implications of the results for retrospective tracking of labelling errors are briefly explored.

An enhanced microsatellite map of diploid Fragaria

A total of 45 microsatellites (SSRs) were developed for mapping in Fragaria. They included 31 newly isolated codominant genomic SSRs from F. nubicola and a further 14 SSRs, derived from an expressed sequence tagged library (EST-SSRs) of the cultivated strawberry, F. × ananassa. These, and an additional 64 previously characterised but unmapped SSRs and EST-SSRs, were scored in the diploid Fragaria interspecific F2 mapping population (FV×FN) derived from a cross between F. vesca 815 and F. nubicola 601. The cosegregation data of these 109 SSRs, and of 73 previously mapped molecular markers, were used to elaborate an enhanced linkage map. The map is composed of 182 molecular markers (175 microsatellites, six gene specific markers and one sequence-characterised amplified region) and spans 424 cM over seven linkage groups. The average marker spacing is 2.3 cM/marker and the map now contains just eight gaps longer than 10 cM. The transferability of the new SSR markers to the cultivated strawberry was demonstrated using eight cultivars. Because of the transferable nature of these markers, the map produced will provide a useful reference framework for the development of linkage maps of the cultivated strawberry and for the development of other key resources for Fragaria such as a physical map. In addition, the map now provides a framework upon which to place transferable markers, such as genes of known function, for comparative mapping purposes within Rosaceae.

The development of ISSR-derived SCAR markers around the Seasonal Flowering Locus (SFL) in fragaria vesca

Fragaria vesca is a short-lived perennial with a seasonal-flowering habit. Seasonality of flowering is widespread in the Rosaceae and is also found in the majority of temperate polycarpic perennials. Genetic analysis has shown that seasonal flowering is controlled by a single gene in F. vesca, the SEASONAL FLOWERING LOCUS (SFL). Here, we report progress towards the marker-assisted selection and positional cloning of SFL, in which three ISSR markers linked to SFL were converted to locus-specific sequence-characterized amplified region (SCAR1–SCAR3) markers to allow large-scale screening of mapping progenies. We believe this is the first study describing the development of SCAR markers from ISSR profiles. The work also provides useful insight into the nature of polymorphisms generated by the ISSR marker system. Our results indicate that the ISSR polymorphisms originally detected were probably caused by point mutations in the positions targeted by primer anchors (causing differential PCR failure), by indels within the amplicon (leading to variation in amplicon size) and by internal sequence differences (leading to variation in DNA folding and so in band mobility). The cause of the original ISSR polymorphism was important in the selection of appropriate strategies for SCAR-marker development. The SCAR markers produced were mapped using a F. vesca f. vesca × F. vesca f. semperflorens testcross population. Marker SCAR2 was inseparable from the SFL, whereas SCAR1 mapped 3.0 cM to the north of the gene and SCAR3 1.7 cM to its south.

A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria

Diploid Fragaria provide a potential model for genomic studies in the Rosaceae. To develop a genetic linkage map of diploid Fragaria, we scored 78 markers (68 microsatellites, one sequence-characterised amplified region, six gene-specific markers and three morphological traits) in an interspecific F2 population of 94 plants generated from a cross of F.vesca f. semperflorens × F. nubicola. Co-segregation analysis arranged 76 markers into seven discrete linkage groups covering 448 cM, with linkage group sizes ranging from 100.3 cM to 22.9 cM. Marker coverage was generally good; however some clustering of markers was observed on six of the seven linkage groups. Segregation distortion was observed at a high proportion of loci (54%), which could reflect the interspecific nature of the progeny and, in some cases, the self-incompatibility of F. nubicola. Such distortion may also account for some of the marker clustering observed in the map. One of the morphological markers, pale-green leaf (pg) has not previously been mapped in Fragaria and was located to the mid-point of linkage group VI. The transferable nature of the markers used in this study means that the map will be ideal for use as a framework for additional marker incorporation aimed at enhancing and resolving map coverage of the diploid Fragaria genome. The map also provides a sound basis for linkage map transfer to the cultivated octoploid strawberry.

Likelihood-based estimation of microsatellite mutation rates

Microsatellites are widely used in genetic analyses, many of which require reliable estimates of microsatellite mutation rates, yet the factors determining mutation rates are uncertain. The most straightforward and conclusive method by which to study mutation is direct observation of allele transmissions in parent-child pairs, and studies of this type suggest a positive, possibly exponential, relationship between mutation rate and allele size, together with a bias toward length increase. Except for microsatellites on the Y chromosome, however, previous analyses have not made full use of available data and may have introduced bias: mutations have been identified only where child genotypes could not be generated by transmission from parents' genotypes, so that the probability that a mutation is detected depends on the distribution of allele lengths and varies with allele length. We introduce a likelihood-based approach that has two key advantages over existing methods. First, we can make formal comparisons between competing models of microsatellite evolution; second, we obtain asymptotically unbiased and efficient parameter estimates. Application to data composed of 118,866 parent-offspring transmissions of AC microsatellites supports the hypothesis that mutation rate increases exponentially with microsatellite length, with a suggestion that contractions become more likely than expansions as length increases. This would lead to a stationary distribution for allele length maintained by mutational balance. There is no evidence that contractions and expansions differ in their step size distributions.

Genetic differentiation between hosts and locations in populations of latent Botrytis cinerea in southern England

This study tested the hypothesis that aggressive, localized infections and asymptomatic systemic infections were caused by distinct specialized groups of Botrytis cinerea, using microsatellite genotypes at nine loci of 243 isolates of B. cinerea obtained from four hosts (strawberry (Fragaria ´ananassa), blackberry (Rubus fruticosus agg.), dandelion, (Taraxacum of®- cinale agg.) and primrose (Primula vulgaris)) in three regions in southern England (in the vicinities of Brighton, Reading and Bath). The populations were extremely variable, with up to 20 alleles per locus and high genic diversity. Each host in each region had a population of B. cinerea with distinctive genetic features, and there were also consistent host and regional distinctions. The B. cinerea population from strawberry was distinguished from that on other hosts, including blackberry, most notably by a common 154-bp amplicon at locus 5 (present in 35 of 77 samples) that was rare in isolates from other hosts (9¤166), and by the rarity (3¤77) of a 112-bp allele at locus 7 that was common (58¤166) in isolates from other hosts. There was signi®cant linkage disequilibrium overall within the B. cinerea populations on blackberry and strawberry, but with quite different patterns of association among isolates from the two hosts. No evidence was found for differentiation between populations of B. cinerea from systemically infected hosts and those from locally infected fruits.

Detection of somaclonal variation during cocoa somatic embryogenesis characterised using cleaved amplified polymorphic sequence and the new freeware Artbio

The scarcity and stochastic nature of genetic mutations presents a significant challenge for scientists seeking to characterise de novo mutation frequency at specific loci. Such mutations can be particularly numerous during regeneration of plants from in vitro culture and can undermine the value of germplasm conservation efforts. We used cleaved amplified polymorphic sequence (CAPS) analysis to characterise new mutations amongst a clonal population of cocoa plants regenerated via a somatic embryogenesis protocol used previously for cocoa cryopreservation. Efficacy of the CAPS system for mutation detection was greatly improved after an ‘a priori’ in silico screen of reference target sequences for actual and potential restriction enzyme recognition sites using a new freely available software called Artbio. Artbio surveys known sequences for existing restriction enzyme recognition sites but also identifies all single nucleotide polymorphism (SNP) deviations from such motifs. Using this software, we performed an in silico screen of seven loci for restriction sites and their potential mutant SNP variants that were possible from 21 restriction enzymes. The four most informative locus-enzyme combinations were then used to survey the regenerant populations for de novo mutants. We characterised the pattern of point mutations and, using the outputs of Artbio, calculated the ratio of base substitution in 114 somatic embryo-derived cocoa regenerants originating from two explant genotypes. We found 49 polymorphisms, comprising 26.3% of the samples screened, with an inferred rate of 2.8 × 10−3 substitutions/screened base. This elevated rate is of a similar order of magnitude to previous reports of de novo microsatellite length mutations arising in the crop and suggests caution should be exercised when applying somatic embryogenesis for the conservation of plant germplasm.